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#1 Signal required for transcription
- identification of critical promoter sequences by experiment
-> tests of upstream regions cloned in plasmids - assay RNA production deletions
- common features, conservation etc
--> sequence conservation - TATA
3 classes of promoter with complex structure
Class I - ribosomal RNA (rRNA) genes
- great string of tandem repeats
- relative abundance of rRNA achieved by high density of tx and many genes, not stability
- high level of tx not achieved by stacking up RNA pol 1 on UCE, or high stability of pre-initiation complex, but UBF stimulates escape or clearance of pol from promoter
Class II - mRNA, most snRNA
- Enhancer elements - densely packed with motifs, activity independent of distance and orientation
- Upstream promoter elements (or upstream activator sequences). Constitutive/ basal or regulated - -200
- Core promoter comprises TATA + Inr, or Inr +d/s element. Inefficient on its own; specifies start site:
EG. protein binding DNA sequences - upstream promoter sequence
oestrogen receptor - AGGTCA...TGACCT
Class III: tRNA, 5S RNA, other small RNAs
# 2 Activation of a class II promoter
- combination of proteins bind in various combinations to allow activation
- upstream promoter elements and enhancers comprise a number of short motifs of about 6-12 bp, each recognised specifically by an ACTIVATOR, which have a domain (= self-folding unit of protein, assembly together), that recognises the DNA sequence, and another domains that bind to other proteins
- - > EG. of activators - nuclear receptors
- many respond to binding of steroid hormones - they interact with DNA the zinc fingers domain - a double helix that recognises specific bases
- activated by hydrophobic ligands that cause conformational changes, either enabling the protein to enter the nucleus and dimerize or enabling proteins in a repressor complex on DNA to associate with other tx factors.
---- Homeodomains - another common type of DNA-binding domain found in activators
- the bZIP regions of c-Fos and c-Jun. N termini make base-specific contacts; C termini form a coiled-coil to produce the heterodimer
Activator proteins - tx factors - bind specific sequences in enhancer / UPE
- in inducible genes, these upstream sequences may be kept nucleosome-free - DNase-hypersensitive - by sequence-specific factors, or the activators may not be bound already but in repressor complexes as NRs; in other cases, the proteins bind despite the nucleosomes. Each one that binds improves the binding of another one.
